Known microscopes, for example stereomicroscopes having optical zooms, are often configured with means for physically limiting the ray bundle and thus for setting the pupil diameter or numerical aperture. Fixed or variable aperture diaphragms, e.g. iris diaphragms or LCD diaphragms, and/or suitable beam limiters on lenses and lens mounts, can be provided, for example, for this purpose.
The present invention relates both to microscopes in which the numerical aperture of one or more optical channels is variably adjustable by means of suitable aperture diaphragms, and to microscopes that comprise two or more separate optical channels each having a fixed aperture diaphragm. Both cases relate to microscope systems that comprise means for furnishing microscopic images at different numerical apertures.
The numerical aperture determines three essential parameters of an optical image, namely the resolution, brightness, and depth of field.
The maximum resolution capability of a microscope in the focal plane of the objective is limited by light diffraction, which in turn is determined by the numerical aperture of the imaging system. The maximum resolution capability R, indicated in line pairs per millimeter (LP/mm), is proportional to the numerical aperture nA. In simplified fashion, R=3000×nA. High resolution therefore requires a high numerical aperture.
At the same time, however, depth of field—i.e. the sharpness of object regions located outside the focal plane—is also important when viewing objects that are not entirely flat. For geometric reasons, this decreases with increasing numerical aperture and is inversely proportional to numerical aperture. For visual examination, the depth of field (DOF) is described by the empirical Berek formula, according to which DOF=λ/(2×nA2)+0.34/(Mtot×nA), where λ is the wavelength of the light and Mtot the total visual magnification. The wavelength λ and depth of field DOF are indicated, for example, in mm. A high degree of sharpness in image regions above or below the focal plane is accordingly achieved with decreased numerical apertures.
The captured light cone determines the brightness I. In simplified fashion, I=c×nA2, where c represents a constant. High image brightness is thus once again achieved with a high numerical aperture. With digital image capture in particular, however, image brightness can be flexibly adjusted and adapted by way of not only the numerical aperture but also the exposure time, electrical gain, optical filters, or digital post-processing. These adaptations are known to one skilled in the art and will therefore not be explained further.
With conventional microscopes, however, conflicting aims exist at least with regard to resolution and depth of field.
DE 10 2006 036 300 B4 and DE 10 2006 036 768 B4 disclose stereomicroscopes, respectively of the telescope and Greenough type, which present to the observer an image pair whose individual images are resolved with different quality (hereinafter also referred to as “asymmetrical” stereomicroscopes). One of the individual images has a higher numerical aperture and therefore a higher resolution in the focal plane, while the other individual image, having a lower numerical aperture, offers better depth of field. These images are received simultaneously by the observer's two eyes. The image pairs are assembled by the brain in such a way that the observer perceives the respectively better resolved detail from the two images. A physiological phenomenon in the context of image fusion in the human brain is thus exploited.
Digital image acquisition devices do not perform this image fusion, and utilize only one image acquired at fixed aperture. They usually capture exactly one image, based on one beam geometry and the resolution and depth of field defined thereby. The sharpness regions of images acquired by such digital image acquisition devices, i.e. the regions in which a corresponding object is sharply imaged, are therefore considerably more narrowly delimited. As compared with a visual stereoscopic image impression through a stereomicroscope, this is not satisfactory. The disadvantage of digital images as compared with visual observations is moreover compounded by the ability of the human eye to accommodate by up to +/−5 diopters.
The existing art in conventional wide field microscopes (not having stereoscopic beam paths) addresses these problems by so-called “Z stacking,” in which the focus position of the microscope is shifted in steps during acquisition of an image sequence. This requires, however, either moving the specimen stage that has the object, or moving the microscope with respect to the object. In either case a considerable mass must be moved (microscope on Z drive, or specimen stage), which makes the operation complex in terms of both apparatus and time. The time outlay in turn prevents implementation as a live image, which is disadvantageous especially in the context of a movement by the user in an X, Y, and Z direction, and for the observation of living cells.
In addition, under (normally) non-telecentric image acquisition conditions, the image scale of the object changes as the focus position changes. The images acquired by Z stacking therefore do not superimpose exactly onto one another because of the variation in image scale, and must be adapted to one another by correlation or by compression or expansion. This operation involves uncertainties and possible errors in the image to be assembled.
The object of the invention, especially in view of the strong trend toward digital microscopy that is apparent nowadays, is to overcome the aforementioned disadvantages and to enable improved image capture.